Throughout this application, various publications are referenced by Arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein.
Malignancy associated hypercalcemia condition (MAHC) was first described in a patient with a renal carcinoma who had a single bone metastases and developed hypercalcemia and hypophosphatemia (1). It was noted that bone resorption could not readily account for hypophosphatemia and suggested that the tumor might be secreting a humoral factor similar to parathyroid hormone (PTH), with skeletal and renal actions which would produce hypophosphatemia and hypercalcemia. The hypothesis that MAHC could have a humoral basis was supported by the observation that in certain patients without bone metastases, hypercalcemia could be cured by resection of the primary tumor which was distant from the bone (2,3). These patients characteristically have squamous, renal, bladder or ovarian carcinomas and have little or no evidence of skeletal disease.
More detailed clinical evaluations of patients with MAHC (4-6) have indicated that such patients fall into two groups. One group has suppressed nephrogenous cAMP (NcAMP) levels and the other has elevated levels. The former group of patients most commonly have malignancies of the breast or hematologic malignancies in addition to widespread bone metastases suggesting that MAHC may be due to the interaction between the metastases and bone, in this case.
The latter group of patients (elevated NcAMP) exhibit a number of common features including:
1. predominantly renal or squamous carcinomas; PA1 2. few or no bone metastases; PA1 3. significantly elevated excretion of calcium relative to patients with primary hyperparathyroidism; PA1 4. low levels of circulating 1,25-dihydroxyvitamin D [1,25(OH).sub.2 D]; PA1 5. reduced or undetectable levels of circulating immunoreactive parathyroid hormone (PTH); and PA1 6. hypophosphatemia and depressed renal phosphorous thresh hold. PA1 1. stimulate the PTH-receptor linked adenylate cyclase in canine renal cortex membranes; PA1 2. stimulate the PTH-receptor linked adenylate cyclase in rat osteosarcoma cells; PA1 3. bind the PTH-receptor in various tissues with equal affinity as PTH although the efficiency of coupling to adenylate cyclase, and hence the PTHLP stimulated activity relative to the PTH stimulated activity, differs in membranes from different sources; PA1 4. stimulate the resorption of calcium from cultured rat fetal long bones; PA1 5. induce in vivo hypercalcemia when infused into rats or injected into mice; PA1 6. stimulate in vivo 1,25-dihydroxy vitamin D.sub.3 formation when infused into rats; PA1 7. stimulate the excretion of cAMP and phosphorous from perfused rat kidneys; and PA1 8. stimulate migration of rat osteoclasts to cortical and trabecular bone surfaces with equal potency to that of bovine (b) PTH(1-34).
These symptoms indicate a humoral basis for MAHC observed among this group of patients and are presently used to define the syndrome termed humoral hypercalcemia of malignancy (HHM).
Biochemical analyses of tumor extracts from patients with HHM led to the identification of a polypeptide factor capable of stimulating receptor-linked enzymatic activities which are also stimulated by parathyroid hormone (PTH) (7). Purification and N-terminal sequence analysis of this PTH-like protein (PTHLP) revealed a high degree of homology to PTH near the amino-terminus (8 out 13 amino acids) but no homology beyond position 13 (8,9). During the same period of time similar factors were identified in conditioned media from cultured cells derived from patients with HHM, renal carcinomas, a lung carcinoma, and more recently, from an HTLV-1 infected T-cell line (10-17). Purification and amino-terminal sequence analyses of these factors (14,17) indicated amino-termini identical to that of PTHLP purified directly from tumors of HHM patients.
Using oligonucleotide probes based upon the amino-terminal amino acid sequence to screen human cDNA libraries, two cDNA sequences have been obtained encoding identical polypeptides containing a 36 amino acid leader sequence and a 141 amino acid mature protein (18,19). Using similar techniques and a probe based upon the reported cDNA sequence, a third cDNA was obtained which was identical except at the extreme 3' end, where alternative RNA splicing led to an mRNA encoding a 139 amino acid mature protein (20). These results are in agreement with those of others (21) who have also observed only a single copy of the PTHLP gene but 3 different mRNAs, arising from 3 alternative 3'-region splices, encoding mature polypeptides of 139, 141, and 161 amino acids.
Since the determination of the amino acid sequence of PTHLP there have been numerous reports describing the in vitro and in vivo biological activities of synthetic amino-terminal peptides of PTHLP corresponding to the receptor binding and biologically active region of PTH (22-27). The results of these studies indicate that these peptides have the following biological activities:
These activities are expected for a putative factor responsible for HHM. Furthermore, the cloning and expression in bacteria of full length (141 amino acids) PTHLP has recently been accomplished (28). Although the polypeptide was not purified to homogeneity, activity measurements suggested a similar ability to stimulate adenylate cyclase as that of synthetic PTHLP(1-34) (28).
It is not yet clear how the similarities and differences in activities of PTHLP and PTH reflect upon the physiological and pathophysiological role of PTHLP in the regulation of calcium homeostasis. In addition, the importance of sequence differences in the carboxyl-terminal region of mature PTHLP polypeptides resulting from alternative splicing of mRNA has not yet been addressed. One reason for this is that there is no method available for adequate differentiation between PTH and PTHLP in crude systems such as tissue and tumor extracts or blood. An immunological assay would overcome the inherent ambiguity of biological assays by avoiding the involvement of a hormone receptor recognizing both PTH and PTHLP. One type of immuoassay has been reported (29) which involves a rabbit polyclonal antisera recognizing a synthetic amino-terminal peptide of PTHLP. This assay is subject to adverse effects from serum and thus requires an extraction procedure before samples can be tested. Since the circulating form(s) of PTHLP are not known and the physiochemical properties of larger PTHLP species are substantially different than those of the amino-terminal peptide, the extraction procedures currently in use are not likely to yield the correct amounts of PTHLP. Thus, results obtained with this assay have not indicated significant elevation of levels of PTHLP in MAHC patients.
To overcome these problems high affinity monoclonal and polyclonal antibodies which are extremely specific for PTHLP need to be developed. The claims and methods provided below pertain to antibodies with these properties.